Osteoporosis due to aging, steroid treatment or immobility due to injury is an increasing problem within the Veteran population. Osteoporosis, often caused by increased osteoclastic bone resporption, is also associated with increased adipocytes and decreased osteoblasts in the bone marrow. The relative proportion of each cell type is determined by factors that regulate lineage determination of the marrow progenitor cell they share in common. We have exciting new observations that LMP-1, an intracellular protein discovered in our laboratory, inhibits the adipogenic and stimulates the osteogenic pathways in human Mesenchymal Stem Cells (hMSCs). This proposal will demonstrate that LMP-1 exerts these effects by blocking Smurf1 action, and in the final translational aim, will provide proof of concept of a novel anabolic therapeutic strategy for treating osteoporosis using a small molecule developed in our laboratory. RESEARCH PLAN: Our preliminary data suggest that LMP-1 exerts effects on lineage commitment by rapidly enhancing beta-catenin activity. Wnt10b is a secreted factor that binds surface receptors and activates beta- catenin; our preliminary studies show that Wnt10b activity is required for LMP-1 to activate beta-catenin. Under adipogenic culture conditions, we have determined that TNF-alpha activity is also required for LMP-1 activation of beta-catenin. This proposal seeks to unveil the role of each of these molecules in LMP-1-induced events favoring commitment of MSCs to the osteoblast lineage. We will demonstrate that 1) activation of beta- catenin is central to the mechanism by which LMP-1 inhibits adipogenesis and induces the osteoblast lineage commitment in MSCs, 2) that Smurf1 inhibition leads to increased expression of Msx2 via different pathways under adipogenic and osteogenic culture conditions, and 3) that altering lineage commitment in MSCs in vivo has an anabolic effect on bone homeostasis in osteoporotic animals. METHODS: Osteogenesis will be assessed by measuring expression of markers of mature osteoblasts and counting bone nodules; adipogenesis will be assessed by measuring expression of adipocyte markers and lipid accumulation within cultures. Activation of beta-catenin (TCF/LEF), NFkB, or PPARgamma transcriptional activity will be measured using reporter constructs. The necessity for a) enhanced activation of beta-catenin for the effect of LMP-1 on osteogenesis and adipogenesis will be tested by blocking beta-catenin activity with a constituitively active GSK3beta construct; b) increased expression of Msx2 and Wnt10b will be tested by applying siRNA to silence their expression. To probe the initial LMP-1 interaction responsible for these changes, we will apply siRNA to Smurf1 under adipogenic and osteogenic growth conditions and also apply an LMP-1 mimetic small molecule that blocks Smurf1 action. LMP-1 interaction with Smurf1 is predicted to inhibit degradation of signaling molecules including Smad1, Smad5, Traf2, making them more available to mediate events leading to enhanced expression of Wnt10b. We will validate the small molecule designed to mimic the LMP-1 blocking effect on Smurf1 by showing that it increases levels of the target proteins and stabilizes beta- catenin by increasing the expression of Wnt10b. In the final translational aim, we will demonstrate that the Smurf1-blocking small molecule drives commitment of MSCs to the osteoblast lineage in vivo and can alter bone homeostasis in osteoporotic rats by favoring increased bone formation and decreased adipogenesis.